Progression through the cell cycle is the result of a highly regulated series of events in four defined phases. In the DNA synthesis phase (the S phase) the cell duplicates its DNA, which is followed by a gap phase (the G2 phase) in which the cell grows and prepares itself for mitosis. During mitosis (M phase) the cell divides its DNA and all of its contents into two separate daughter cells. Another gap phase (the G1 phase) follows mitosis and precedes the S phase. During the G1 phase cellular components are checked for repair, including DNA, prior to progressing into S phase. The majority of variability in the timing of the cell cycle is accounted for by differences in the rate of passage through G1. Progression though G1 is primarily dependent upon receiving and processing external and internal signals, as well as monitoring of cellular activity. The other cell cycle phases are generally autonomously programmed events, which while normally insulated from extracellular growth signals, are still susceptible to the misregulation of proteins disrupting the checkpoints within the cell cycle phase.
Since cell duplication relies on accurate chromosome separation, mitosis is thought to be one of the most tightly regulated cellular processes. Cells have solved the fidelity problem in part by relying on a sequential processing strategy that involves timing of individual cell cycle steps and on checkpoints that monitor the completion of different cell cycle phases (Ekholm et al., Mol. Cell. Biol. 21, 3256-65 (2001); Elledge et al., Science 274, 1664-72 (1996)). For example, during mitosis, the spindle checkpoint ensures the correct alignment and spindle attachment of sister chromatids (Musacchio et al., Nature Rev. Mol. Cell Biol. 3, 731-41 (2002); Yu, Curr. Opin. Cell Biol. 14, 706-714 (2002)). Failures at this critical checkpoint lead to genetic instability, which is a hallmark of cancer and is correlated with aggressive tumor behavior (e.g., unregulated cellular proliferation).
Techniques such as immunohistochemistry and FACS analysis have provided valuable information for understanding cell-cycle events. However, since these methods provide snapshots of single cells that are usually synchronized (e.g., arrested at the same cell cycle phase prior to analysis), temporal resolution of specific cell cycle phases (e.g., mitosis) at the single cell level is difficult. Phase contrast, differential interference contrast (DIC) or fluorescence imaging of live mitotic cells eliminates some of these problems, and has been used to measure mitotic times of individual cells. Nevertheless, observing mitotic timing in statistically significant cell numbers remains a challenge.
Accordingly, there remains a need in this art for systems and methods for monitoring cell cycle events, including methods for screening agents for use in treating cell cycle associated diseases, such as cancer. The present invention addresses this need.